RNA polymerase is the principal enzyme of gene expression and the target for genetic regulation. Its basic structure-functional features are highly conserved among all living organisms. The broad goal of this project is the understanding of the molecular mechanism of RNA polymerase basic function using site directed mutagenesis of evolutionarily invariant amino acids in individual subunits of RNA polymerase from Escherichia coli. For this purpose, single amino acid substitution mutations, linker insertions and in-frame deletions will be generated in the cloned gene rpoB which encodes the core RNA polymerase beta subunit. The mutant RNA polymerase holoenzyme (alpha-2-beta-beta-'sigma) will then be prepared for biochemical characterization by in vitro reconstitution from the individual subunits. The subunits will be obtained by overexpressing them from plasmids carrying the three rpo genes under the control of inducible promoter. The malfunctioning of RNA polymerase caused by the amino acid substitutions will be characterized using an arsenal of in vitro assays which would allow to monitor and quantitate individual steps of the enzyme's functional cycle. At the second stage of this project, compensatory suppressor mutations will be isolated and mapped. This approach is anticipated to provide information about molecular details of promoter binding, the functioning of the enzyme's catalytic center, the nature of processivity, the mechanism of pausing and termination, as well as to, uncover structural elements of the enzyme which carry out these functions.